A number of novel lipopeptides have been studied for their possible therapeutic potential. These studies should be supported by the appropriate analytical tools not only for novel potential drugs but also for their metabolites, precursors and side products. Lipopeptides have specific physicochemical properties that make them successful in medical applications. However, there are some difficulties with their qualitative and quantitative analyses in biological samples. Therefore, reliable, sensitive and robust analytical methods are in high demand. The main interest of our review is to describe a selection of specific and important properties of lipopeptides, and the analytical methods currently utilized for their characterization and determination in biological samples. A comparison of the pros and cons of immunomethods versus LC–MS methods is discussed in detail.Trvalý link: http://hdl.handle.net/11104/0271124